J . Am. Chem. SOC.1986, 108, 5309-5316
5309
P-Hydroxydecanoyl Thioester Dehydrase. Complete Characterization of the Fate of the “Suicide” Substrate 3-Decynoyl-NAC John M. Scbwab,*1.2Chorng-Kei Ho,* Wu-bo Li,2 Craig A. T ~ w n s e n d ,and ~,~ Gino M. Salituro3 Contribution from the Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907, Department of Chemistry, The Catholic University of America, Washington, D.C. 20064, and Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21 218. Received December 16. 1985
Abstract: j3-Hydroxydecanoyl thioester dehydrase, the pivotal enzyme in the biosynthesis of unsaturated fatty acids under anaerobic conditions, catalyzes the interconversion of thioesters of (R)-3-hydroxydecanoic acid (l), (E)-2-decenoic acid (2), and (Z)-3-decenoic acid (3). Dehydrase is irreversibly inactivated by the N-acetylcysteamine thioester of 3-decynoic acid (3-decynoyl-NAC), via dehydrasecatalyzed conversion of the acetylenic thioester to 2,3-decadienoyl-NAC. This is the classic example of “suicide” or “mechanism-based” enzyme inactivation. NMR-based experiments have been carried out in order to define the mechanistic relationship between “normal” catalysis and suicide inactivation of dehydrase, by providing detailed structural information on the enzyme-bound inactivator moiety. 3-[2-13C]Decynoyl-NACwas synthesized and incubated with homogeneous dehydrase. 13CN M R spectroscopy at 100.6 MHz showed that when 2,3-decadienoyl-NAC is attacked by the active-site histidine, the product is (3-imidazolyl-3-decenoyl)-NAC. This adduct is slowly isomerized to (3-imidazolyl-2decenoy1)-NAC. One molecule of inactivator is bound per subunit of the dimeric enzyme. Model histidine-allene adducts have been made and characterized. Comparisons of NMR data reveal that the double-bond configuration of the decenoyl moiety of the enzyme-bound inactivator is E. Analysis of these findings strongly suggests that the histidine residue that is alkylated by 2,3-decadienoyl-NAC is the active-site base. The structure of the product formed by inactivation of dehydrase by 3-decynoyl-NAC and the mechanism of the inactivation are readily explained in terms of the mechanisms of the normal dehydrase-catalyzed reactions as well as the stereochemical relationships between enzyme and substrates in those normal reactions.
8-Hydroxydecanoyl thioester d e h y d r a ~ e the , ~ pivotal enzyme in the biosynthesis of unsaturated fatty acids under anaerobic conditions, catalyzes the interconversion of thioesters of (R)-3hydroxydecanoic acid (1), (E)-2-decenoic acid (Z), and (2)-3decenoic acid (3) (Scheme I). While clearly important metabolically, dehydrase is perhaps better known for the fact that it is rapidly and completely inactivated by incubation with the N-acetylcysteamine (NAC) thioester of 3-decynoic This experimental observation constituted the original demonstration of “suicide” enzyme inactivation,8 a phenomenon often referred to as “mechanism-based“ enzyme inactivation. Unfortunately, the mechanistic bases for the action of mechanism-based inactivators have, until very re~ e n t l y , ” ~always been inferred rather than proven unequivocally through carefully designed experiments. Such has been the case for the inactivation of dehydrase by 3-decynoyl-NAC. Over the past several years we have sought a better understanding of both the “normal” dehydrase-catalyzed reactions (equilibration of 1, 2, and 3) and the inactivation of the enzyme by 3-decynoyl-NAC, in order to better define the mechanistic relationship between these processes.
Scheme I. Reactions Catalyzed by 8-Hydroxydecanoyl Thioester
Dehydrase
1
2
3
Scheme 11. Possible Partial Structures for the Dehydrase-Linked
lnactivator
Scheme 111. Synthesis of 3-[2-13C]Decynoyl-NAC C6Ht3-CX-H
1 EtMgBr
C6H13-CiC-CH20H
2 %H,O
(1) To whom correspondence should be addressed at Purdue University.
0002-7863/86/1508-5309$01.50/0
-
CuCN/LiBr/OMF
-
C6Hl3-CiC-CH2CN
-
1. MeOH/HCI
2 . dil. NaOH
5L%
1. PhOPOCI,/Et,N
C,H,,-C~C-EH,B~ 70%
66%
(2) The Catholic University of America.
(3) The Johns Hopkins University. (4) Research Fellow of the Alfred P.Sloan Foundation (1982-1984) and Camille and Henry Dreyfus Teacher-Scholar (1983-1988). (5) Bloch, K. In The Emymes, 3rd ed.;Boyer, P.D., Ed.;Academic: New York, 1971; Vol. 5, pp 441-464. (6) Brock, D. J. H.;Kass, L. R.; Bloch, K. J. Biol. Chem. 1967, 242, 4432-4440. (7) Kass, L. R.;Bloch, K. Proc. Natl. Acad. Sei. U.S.A. 1967, 58, 1168-1173. (8) Walsh, C. Tetrahedron 1982, 38, 871-909. (9) Schwab, J. M.;Li, W.-b.; Ho, C.-K.; Townsend, C. A,; Salituro, G . M.J . Am. Chem. SOC.1984, 106, 7293-7294. (10) Badet, B.; Roise, D.; Walsh, C. T. Biochemistry 1984,23,5188-5194. (11) Roise, D.; Soda, K.; Yagi, T.; Walsh, C. T. Biochemistry 1984, 23, 5195-5201. (121 Gelb. M. H.: Abeles. R. H. Biochemisrrv 1984. 23. 6596-6604. (13j Ringe, D.; Seaton, B: A.; Gelb, M. H.; Abeles, R.H. Biochemistry 1985.24, 64-68.
PBr3
pyridine
*
CsH13-CiC-CH2-COOH
56%
0 C~H,~--=&CVNHCOCH~
2 TISCHzCH2NHAc
50%
Herein, we describe NMR-based experiments that provide a complete regio- and stereochemical picture of the fate of 3-decynoyl-NAC, following incubation with d e h y d r a ~ e . ~
Results Regiochemistry of the Enzyme-Bound Vinyl Imidazole. The initial step in characterizing the structure of the dehydrase-in0 1986 American Chemical Society
5310
Schwab et al.
J. Am. Chem. SOC.,Vol. 108, No. 17, 1986
OD210 110
-I
n
3000 -
2000 1000
-
dpm
Figure 1. I3C(IHJNMR spectra, obtained on a Varian XL-400, under conditions described in the Experimental Section: (a) dehydrase (51 mg, 1.42 pmol), 75000 scans; (b) dehydrase in (a), but after addition of 0.34 mg (1.26 pmol) of 3-[2-13C]decynoyl-NAC;(c) dehydrase in (b), but after 2-week storage in a refrigerator at 4 OC.
activator adduct is to determine the position of the carbon-carbon double bond in the inactivator moiety. Specifically, attack of the active-site histidine a t C-3 of the allenic thioester will lead to a thioester dienolate (or dienol) that could in principle be protonated either a t C-2 or C-4 (Scheme 11). Protonation a t C-2 would give a nonconjugated vinyl imidazole thioester, while protonation a t C-4 would afford the conjugated species. While a chemical degradation approach had been used previously to access this point,I4 the experiments were arduous, and the results were not unequivocal. A carbon-13 labeling experiment (using I3CN M R for product analysis) was therefore designed. 3-[2-13C]Decynoic acid was synthesized (Scheme 111) from ["Clformaldehyde by a modification of the route used by HelmkampI5 for the synthesis of 3-[l-14C]decynoic acid. The acid was thioesterified by conversion to a mixed anhydride,I6 followed by treatment of the latter with the thallium salt of NAC.17,'8 Homogeneous dehydrase was prepared from the cloned, overproducing mutant Escherichia coli DM5 1-A by straightforward methods, based largely on the prior work of HelmkampI5 and Kass et al.19 Since protein concentration determinations proved to be strongly dependent on the nature of the protein standard, gravimetric analyses were performed on aliquots of dehydrase. In this way, the quantities of protein used in subsequent N M R experiments could be accurately known. A 100.6-MHz 13C{lH]N M R spectrum was obtained (Figure l a ) on 51 mg of dehydrase. By use of WALTZ-16 proton decoupling,20 the sample temperature reached only 27 OC, and so no external cooling was required to prevent denaturation. In fact, (14) Stein, J. P. Ph.D. Thesis, Harvard University, Cambridge, MA, 1970. (15) Helmkamp, G. M., Jr. Ph.D. Thesis, Harvard University, Cambridge, MA, 1970. (16) Liu, H.-J.; Sabesan, S.I. Can. J . Chem. 1980, 58, 2645-2648. (17) Schwab, J. M.; Klassen, J. E. J. Am. Chem. Soc. 1984, 106, 7217-7227. (18) Masamune, S.; Kamata, S.; Diakur, J.; Sugihara, Y.; Bates, G. S. Can. J . Chem. 1915, 53, 3693-3695. (19) Kass, L. R.; Brock, D. J. H.; Bloch, K. J . Biol. Chem. 1967, 242, 4418-443 1. (20) Shaka, A. J.; Keeler, J.; Freeman, R. J. Magn. Reson. 1983, 53, 3 13-340.
Figure 2. Reversed-phase HPLC profile of the hydrolysate from dehydrase inactivated with 3-[l-'4C]dccynoyl-NAC. Both UV and radioactivity profiles are shown. The chromatographic conditions are described in the Experimental Section.
Scheme IV. Synthesis of Model Adducts
OASR
6
5
only 15% of the original enzyme activity was lost over the course of a 17-h data acquisition. A substoichiometric quantity of 3[2-'3C]decynoyl-NAC was then added and a second spectrum obtained (Figure lb). Two new resonances are evident, a t 45.0 and 110.2 ppm, with a ratio of integrals of roughly 2:l. The line widths (36 and 28 Hz, respectively) indicate that these signals owe to enzyme-bound inactivator. (Dehydrase is a dimeric enzyme, with a subunit molecular weight of 18000.) Significantly, it was found that aged, inactivated enzyme (2 weeks at 4 "C) gave the spectrum shown in Figure IC, exhibiting a reversal of the ratio of integrals, such that the 110.2 ppm peak became predominant. This, plus the fact that early in data acquisition only the 45 ppm signal was seen (the 110 ppm signal was imperceptible), suggests that the species responsible for the 45 ppm peak is formed rapidly and that there is a slow, subsequent conversion of it to the species with the C-2 chemical shift of 110 ppm. The assignment of structures to the moieties with the 45 and 110 ppm C-2chemical shifts rests on comparisons to model compounds. As had been found by Morisaki and Bloch?' and as shown in Scheme IV, the nonconjugated vinyl imidazole thioester (tentatively assigned structure 4; vide infra) is formed in the reaction between N-acetyl-L-histidine methyl ester and 2,3-decadienoic acid, n-propyl thioester (Morisaki had used the ethyl thioester). Carbon-2 of 4 resonates a t 45.1 ppm. When (21) Morisaki, M.; Bloch, K. Biochemisfry 1972, 11, 309-314.
J . Am. Chem. Soc., Vol. 108, No. 17, 1986 5311
Suicide Inactivation of the Dehydrase
Table I. Spectral Characteristics of Adducts Formed between N-Acetyl-L-histidine Methyl Ester and 2,3-[2-"C] Decadienethioic Acid, S-1-Propyl Ester reactants DI'odUCt compd amt recovered, ma 3Jru.Hz c-2 6 CHsOC,
YNYC00CH3 25
7.0
45.1
10
7.0
46.1
c8H,sa
